Hybrid nanoparticles improve targeting to inflammatory macrophages through phagocytic signals

Abstract

Macrophages are innate immune cells with great phenotypic plasticity, which allows them to regulate an array of physiological processes such as host defense, tissue repair, and lipid/lipoprotein metabolism. In this proof-of-principle study, we report that macrophages of the M1 inflammatory phenotype can be selectively targeted by model hybrid lipid-latex (LiLa) nanoparticles bearing phagocytic signals. We demonstrate a simple and robust route to fabricate nanoparticles and then show their efficacy through imaging and drug delivery in inflammatory disease models of atherosclerosis and obesity. Self-assembled LiLa nanoparticles can be modified with a variety of hydrophobic entities such as drug cargos, signaling lipids, and imaging reporters resulting in sub-100nm nanoparticles with low polydispersities. The optimized theranostic LiLa formulation with gadolinium, fluorescein and "eat-me" phagocytic signals (Gd-FITC-LiLa) a) demonstrates high relaxivity that improves magnetic resonance imaging (MRI) sensitivity, b) encapsulates hydrophobic drugs at up to 60% by weight, and c) selectively targets inflammatory M1 macrophages concomitant with controlled release of the payload of anti-inflammatory drug. The mechanism and kinetics of the payload discharge appeared to be phospholipase A2 activity-dependent, as determined by means of intracellular Förster resonance energy transfer (FRET). In vivo, LiLa targets M1 macrophages in a mouse model of atherosclerosis, allowing noninvasive imaging of atherosclerotic plaque by MRI. In the context of obesity, LiLa particles were selectively deposited to M1 macrophages within inflamed adipose tissue, as demonstrated by single-photon intravital imaging in mice. Collectively, our results suggest that phagocytic signals can preferentially target inflammatory macrophages in experimental models of atherosclerosis and obesity, thus opening the possibility of future clinical applications that diagnose/treat these conditions. Tunable LiLa nanoparticles reported here can serve as a model theranostic platform with application in various types of imaging of the diseases such as cardiovascular disorders, obesity, and cancer where macrophages play a pathogenic role.

Keywords: Atherosclerosis; DOPSE (PubChem CID: 6438639); Gd-DTPA (PubChem CID: 55466); Inflammation; Intravital imaging; Magnetic resonance imaging; Obesity; PEG2000 DSPE (PubChem CID: 406952); Paclitaxel (PubChem CID: 36314); Rosiglitazone (PubChem CID: 77999); Tamoxifen (PubChem CID: 2733526); Theranostic nanoparticles.

Fig. 1

Schematic illustration of components in LiLa nanoparticles. The single step self-assembly with a latex core template, lipids, dyes/drugs yields theranostic lipid–latex hybrid nanoparticles (LiLa). The targeting to macrophages is achieved by phagocytic signals phosphatidylserine (PtdSer) and cholesterol-9-carboxynonanoate (9-CCN). LiLa formulations bearing Gd or Alexa-647 imaging probes are expected to serve as contrast agents for MRI and fluorescence imaging.

Fig. 2

Physicochemical, magnetic and drug-loading properties of LiLa. (a) A relationship between the concentration of the lipid fraction in LiLa, nanoparticles' size and dispersity. The hydrodynamic size and polydispersity index (PDI) was measured by DLS for 12 formulations and presented as mean of three replicates ± standard error on double Y-axis (red, left is size and right, blue is PDI). (b) The cryo-TEM micrographs of Gd-LiLa nanoparticles. Yellow arrows indicate the electron-dense spots attributed to Gd deposition on the surface of the latex. White arrows depict prominent PEG corona around the particles. (c) MRI relaxivity characteristics of Gd-LiLa nanoparticles presented as a function of the relaxation times T₁ vs gadolinium concentration. T₁ values were extracted from the signal intensity of the corresponding phantoms (inset) after curve fitting using a range of repetition times (see methods). The molar relaxivity, r₁, was obtained from the slope of ${\mathrm{T}}_1^{-1}$ vs gadolinium concentration. (d) Drug loading capacity of LiLa as determined on three model hydrophobic drugs: rosiglitazone (Rosi), paclitaxel (PAX) and tamoxifen (TAM). The data obtained from three different formulations for each drug is presented as mean ± standard error. (e) Plasma stability of Rosi-LiLa as determined by dynamic dialysis against 50% human plasma at 37 °C.

Fig. 3

In vitro characterization of various LiLa nanoparticles in different cell lines. (a) Confocal microscopy was used to evaluate Gd-FITC-LiLa (green) uptake in fibroblasts (L-cells), human umbilical vein endothelial cells (HUVEC) and macrophages (RAW 264.7). The right panel represents the overlay of fluorescent and bright field (DIC) micrographs. For visualization of the lysosomal compartment, cells were stained with antibody against lysosomal-associated membrane protein 1 (red). (b) Time-dependent uptake of Gd-LiLa by different cell lines. The data were normalized to the protein content in cell lysates. The quantification was based on 3–4 independent replicates, and the error bars represent standard deviation. The statistical analysis of the time-course trend by two-way ANOVA demonstrated a significant difference in uptake of Gd-LiLa by RAW cells as compared to HUVEC (p < 0.001) and L-cells (p < 0.01). (c,d) Flow cytometry analysis of bone marrow derived macrophages that were polarized to M1 and M2 phenotypes and treated with bare latex or LiLa nanoparticles. The M1 inflammatory phenotype showed higher uptake of LiLa particles compared to M2 phenotype. Single-cell analysis by means of imaging flow-cytometry (inset) suggested intracellular localization of LiLa in M1-macrophages (see text).

Fig. 4

Therapeutic properties of rosiglitazone-loaded LiLa (Rosi-LiLa) in an in vitro model of inflammation. (a, b) An in vitro model of inflammation was created and evaluated by flow-cytometry using a marker of inflammation lymphocyte antigen 6C (Ly6C) and a macrophage-specific antibody F4/80. A treatment of RAW 264.7 macrophages with lipopolysaccharide (LPS) gave rise to inflammatory macrophages, of which 18% were Ly6C-positive. The control macrophages (buffer-treated) were only 2.5%-positive for Ly6C. (c, d) Antiinflammatory effects of Rosi-LiLa were studied in the inflammation model described above. A treatment of inflammatory macrophages with Rosi-LiLa resulted in significant reduction of Th1 response with significantly attenuated production of indicated cytokines (c) and mRNA transcripts (d). The data are expressed on a logarithmic scale as percent change in cytokine concentration over concentration in cells treated with phosphate buffered saline (PBS). The error bars denote standard deviation of three separate experiments performed in triplicate. t-Test was used to determine significance. *p < 0.05, †p < 0.01 vs. PBS control.

Fig. 5

Controlled release of the model drug from LiLa nanoparticles. (a) Design of FRET-LiLa beacon: fluorescein incorporated latex core (green) is decorated with rhodamine (red) producing a FRET pair with excitation λ₁ = 460 nm and emission λ₃ = 550 nm. Upon cleavage of phospholipid layer (yellow) with methanol or phospholipase PLA2, rhodamine is released resulting in a FRET loss (fluorescein emitting at λ₂ = 480 nm). Inset:schematic representation of formation of LysoPC, the product of PLA2-induced phospholipid hydrolysis. (b) Fluorescence emission spectra of FRET-LiLa in water (red curve) and methanol (blue curve). (c) FRET-loss was monitored by recording changes in λ₂ (480 nm) signal in three LiLa formulations after exposure to PLA2. Snake-venom PLA2 was simultaneously added to all three formulations at 6 min of incubation. (d) Confocal fluorescence images of FRET-LiLa-treated RAW cells in sensitized emission FRET (pFRET) mode before (MAFP−) and after (MAFP+) the treatment with PLA2 inhibitor (MAFP). (e) pFRET image analysis (7–10 slides per group were analyzed) gave rise to FRET efficiency and FRET distance of MAFP treated and untreated cells. A distance ≥10 nm indicates no FRET. (f) Rhodamine fluorescence emission spectra recorded in PBS or 30% human serum over time for latex nanoparticles incorporating rhodamine but no phospholipids (Rhod-Latex), LiLa particles with rhodamine and phospholipids (Rhod-LiLa), and LiLa particles with rhodamine where 50% of phosphatidylcholine was replaced with Lyso-phosphatidylcholine (Rhod-LysoPC-Latex). t-Test was used to determine significance. *p < 0.05.

Fig. 6

In vivo MRI, blood clearance and macrophage targeting characteristics of Gd-FITC-LiLa nanoparticles. (a) Representative time-lapse magnetic resonance imaging (MRI) scans of vasculature in wild type (WT) and atherosclerotic ApoE^−/− knockout (KO) animals following injection of Gd-FITC-LiLa. The early time-point images show high contrast enhancement in abdominal aorta (A), celiac trunk (C), superior mesenteric artery (M), and kidney (K). (b) The Gd-FITC-LiLa blood clearance in WT and KO mice was determined by measuring the concentration of Gd in blood over time by inductively coupled plasma mass spectrometry. The data presented are from seven mice (n = 4 KO and n = 3 WT) and the error represents the standard deviation of biological replicates. (c) A representative T₁-weighted gradient echo MR scan in the axial plane (right) and the corresponding magnifications depicting the aortic wall (arrows) in WT and KO mice before and 24 h after Gd-FITC-LiLa injection. Prominent contrast-enhanced delineation of the aortic wall is clearly seen as bight hyperintense signal in KO mice 24 h post Gd-FITC-LiLa injection. (d, e) Confocal microscopy micrographs of atherosclerotic plaque of KO mice treated with Gd-FITC-LiLa. (d) The accumulation of Gd-FITC-LiLa in shoulders of atherosclerotic plaque (asterisks) was observed in overlaid DIC and fluorescence images at 20× magnification. (e). The signal from F4/80-positive (red) macrophages co-localized with fluorescence of Gd-FITC-LiLa (green) and was seen as yellow on micrographs of atherosclerotic plaque at 60× magnification. Nuclei (blue) were detected through staining with DAPI.

Fig. 7

In vivo confocal imaging with LiLa nanoparticles. (a) Schematic view of a confocal microscope used for intravital imaging of adipose. A laser light is focused through a pinhole array, and then reflected into a galvanometer scanner (galvo) by the dichroic mirror. The laser beam is then used to scan the sample through galvanometer as it moves across the image. The emitted light is then “descanned” back to the galvanometer allowing for synchronized imaging with minimal distortion. A live imaging is performed through a small incision in animal's adipose pad. (b, c) Representative time-lapse confocal images of the adipose tissue in M1 and M2 mouse models of adipose inflammation. “M1” (obese, n = 3) and “M2” (lean, n = 3) yellow fluorescent protein (YFP) reporter mice were injected with AF647-LiLa nanoparticles and subjected to intravital imaging monitoring fluorescence signals from YFP⁺ macrophages (green) and AF647-LiLa (red) over time. (d) Quantification of mean fluorescence intensity of AF647 (expressed as % of maximum fluorescence) in YFP⁺ macrophages over time. Image series were analyzed with ImageJ software by selecting YFP⁺ region of interest in each frame, followed by analysis of average fluorescence intensity of AF647 for each time point (e) Area under the curve (AUC) was calculated to determine the rate of uptake of AF647-LiLa in macrophages. t-Test *p < 0.01 M1-model vs M2-model.